Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disease in the U.S., with an estimated 20 million people displaying evidence of fat-related liver injury. The rapid rise in NAFLD since 1970 suggests that environmental factors play a critical role in the pathogenesis of the disease. Epidemiologic studies point to dietary sugar as a specific risk factor for NAFLD; sugar can harm liver cells by being converted to toxic long- chain saturated fatty acids (SFA) through the process of de novo lipogenesis (DNL). DNL is not the only route by which SFA enter the liver, but studies from our laboratory suggest that DNL-derived SFA are particularly hepatotoxic. In contrast, SFA present in the diet induce relatively little liver injury in vivo. Interestingly, despite their apparently innocuous nature when fed with other nutrients, dietary SFA can synergize with dietary sugar to promote severe fatty liver disease. The hypothesis that forms the basis of this proposal is that DNL SFA are major mediators of hepatocellular injury in NAFLD. Accordingly, dietary nutrients or nutrient combinations that cause significant fatty liver disease likely do so in direct relationship to their ability to stimulate DNL. Specific Aim 1 will investigate the possibility that dietary saturated fat synergizes with dietary sugar to promote fatty liver disease not by contributing directly to a cytotoxic SFA pool, but instead by amplifying DNL and increasing hepatic production of toxic DNL SFA. Experiments will also address the hypothesis that dietary SFA are themselves less toxic than DNL SFA due to unique partitioning within the liver. These experiments will utilize the methionine-choline-deficient model of murine fatty liver disease, in which liver injury is known to depend upon the hepatic accumulation of DNL SFA. Dietary and DNL fatty acids will be individually traced through several metabolic pathways in vivo by labeling with unique stable isotopes. Specific Aim 2 will explore the cellular events by which DNL leads to hepatocyte death. Experiments will confirm that hepatocyte death is directly linked to DNL using pharmacologic and genetic means to induce DNL, and will target various intracellular signaling pathways activated by DNL to determine which ones mediate cell death. Specific Aim 3 will test the hypothesis that DNL SFA are pivotal mediators of fatty liver disease not only in the MCD-fed mouse, but also in a standard, non-MCD model of diet-induced obesity. As in the MCD model, dietary SFA are expected to play a limited role in the pathogenesis of diet-induced liver injury in comparison to DNL SFA. The ultimate goal of the project is to demonstrate the importance of DNL to fatty liver disease and by analogy, the importance of dietary sugar and other nutrients that stimulate DNL. The information gained from this research will guide policymakers to develop nutritional guidelines that minimize excess DNL.